1. Field of the Invention
The present invention relates to reagents and methods for classifying cells, and in particular, to reagents and methods for classifying leukocytes into four groups.
2. Description of the Related Art
In clinical testing fields, it is common and important to classifying and counting leukocytes in the whole blood of patients for diagnosis of diseases.
Till now, a number of devices and methods for classifying and counting leukocytes have been reported.
CN 95115317.X discloses a method for classifying leukocytes into five groups with two kinds of reagents. Both the reagents contain nonionic surfactants and a buffer. In addition, the first reagent for classifying the blood sample into four groups consisting of three groups corresponding to lymphocytes, monocytes and eosinophils and one group corresponding to neutrophils and basophils comprises at least one ionic surfactant, at least one organic compound having an anionic group and a buffer for adjusting pH, while the second reagent comprises at least one ionic surfactant for classifying the basophils from leukocytes other than basophils. By combining the information obtained with both reagents, this method classifies leukocytes into five groups. The detection system is based on laser light scattering. It is also emphasized that the nonionic surfactant is required and leukocytes will be lysed as a side effect which should be prevented.
U.S. Pat. No. 5,618,733 discloses a reagent for classifying and counting leukocytes. Specifically, it discloses a reagent for classifying leucocytes at least into four subgroups, which comprises at least one ionic surfactant, at least one organic compound, a nonionic surfactant and a buffer for adjusting pH. According to this patent, the reagent may comprise a first liquid and a second liquid, wherein said first liquid comprises at least one organic compound and wherein said second liquid comprises at least one ionic surfactant. It is also emphasized therein that each of said first and second liquids comprises a nonionic surfactant and a buffer, and the nonionic surfactant is necessary for dissolving the insoluble substance.
U.S. Pat. No. 5,389,549 relates to a method for classifying leukocytes employing the RF method and the DC method and a reagent used therefor. The reagent comprises a first fluid for diluting blood and which contains a hyperosmotic agent, a second fluid contains an anionic surfactant or a nonionic surfactant.
U.S. Pat. No. 5,538,893 relates to a reagent for classifying and counting leukocytes having at least one nonionic surfactant, at least one cationic surfactant, and a buffer.
Chinese Patent Application No. 88101677 relates to a method and reagent system for isolation and identification of leukocyte subpopulation. Said reagent system comprises a lytic reagent and a quench. The measuring techniques can be a radio frequency (RF) method or a direct current (DC) method.
U.S. Pat. No. 5,155,044 discloses a lytic reagent system for selective chemical treatment of whole blood, which comprises an acidic aqueous solution consisting essentially of a diluent, a lytic reagent and optionally a separate quench. The lytic reagent consists of formic acid, acetic acid and their respective mixtures. The reagent system can contain saponin in addition to the lytic reagent. And the pH value is in the range of from about 6 to about 7.5.
U.S. Pat. No. 5,731,206 discloses a lytic reagent composition and a kit of a lytic reagent system for the rapid isolation, identification and/or analysis of at least five subpopulations of leukocytes from a whole blood sample. The kit of a lytic reagent system comprises a first composition which decreases the pH of the sample to a pH in the range of from about 2.6 to about 4.0 containing saponin and carboxylic or sulfonic acid derivatives, and a second composition comprising a quench which stabilizes the blood sample at a pH in the range of from about 6 to about 7.5.
In U.S. Pat. No. 5,817,518, it discloses a reagent and method for differential determination of leukocytes in blood using DC and RF measurements. The lytic reagent comprises a nonionic surfactant that protects the neutrophil subpopulation from lysis. The pH value of the lytic reagent is adjusted to a pH in the range of from about 1.4 to about 3.4.
U.S. Pat. No. 5,510,267 provides a lytic agent and method enabling 5-part leukocyte differential count, wherein the lytic agent consists essentially of an aromatic oxyethanol, an organic buffer, and a nonionic detergent. The testing system is based on a four-angle laser light diffraction.
Among the above-mentioned prior art reagents and methods for classifying leukocytes, nonionic surfactants are necessary to the reagent for classification of CN95115317.X, U.S. Pat. No. 5,618,733, U.S. Pat. No. 5,389,549 and U.S. Pat. No. 5,538,893. There are also some restrictions on the pH value. Taking CN95115317.X for example, it is required that the pH value should be in the range of 5-11. All of these restrictions prevent the above-mentioned techniques from wide application.
Furthermore, with regard to the techniques for classifying described in CN 88101677, the most suitable condition is at normal temperature. With the changing of the ambient temperature, it is difficult to maintain the temperature constant at 25° C. The measurements fluctuate with the temperature and thus hurt the measurement precision. Under the circumstances, thermostatic apparatus are required to eliminate the influence of temperature changing. In addition, to keep the temperature at 25° C., not only heating devices but also cooling devices are necessary, which significantly increase the equipment costs.
Thus, there continues to be a need for improvement in the art for reagents and methods for classifying leukocytes that do not have the above drawbacks.